Glycoengineering enabled the production of proteins with human N-linked glycans by Pichia pastoris. This study used a glycoengineered P. pastoris strain which is capable of producing humanized glycoprotein with terminal galactose for monoclonal antibody production. A design of experiments approach was used to optimize the process parameters. Followed by further optimization of the specific methanol feed rate, induction duration, and the initial induction biomass, the resulting process yielded up to 1.6 g/L of monoclonal antibody. This process was also scaled-up to 1,200-L scale, and the process profiles, productivity, and product quality were comparable with 30-L scale. The successful scale-up demonstrated that this glycoengineered P. pastoris fermentation process is a robust and commercially viable process.
During early preclinical development of therapeutic proteins, representative materials are often required for process development, such as for pharmacokinetic/pharmacodynamic studies in animals, formulation design, and analytical assay development. To rapidly generate large amounts of representative materials, transient transfection is commonly used. Because of the typical low yields with transient transfection, especially in CHO cells, here we describe an alternative strategy using stable transfection pool technology. Using stable transfection pools, gram quantities of monoclonal antibody (Mab) can be generated within 2 months post-transfection. Expression levels for monoclonal antibodies can be achieved ranging from 100 mg/L to over 1000 mg/L. This methodology was successfully scaled up to a 200 L scale using disposable bioreactor technology for ease of rapid implementation. When fluorescence-activated cell sorting was implemented to enrich the transfection pools for high producers, the productivity could be improved by about three-fold. We also found that an optimal production time window exists to achieve the highest yield because the transfection pools were not stable and productivity generally decreased over length in culture. The introduction of Universal chromatin-opening elements elements into the expression vectors led to significant productivity improvement. The glycan distribution of the Mab product generated from the stable transfection pools was comparable to that from the clonal stable cell lines.
The National Aeronautics and Space Administration (NASA) is currently developing a robotic mission to visit a near-Earth asteroid (NEA) and redirect it into lunar orbit. Once this mission is complete, NASA plans to send a manned mission to the same NEA so that astronauts can explore it and return to Earth with samples from the NEA's surface. Doing so will require a new set of extravehicular activity (EVA) tools, as existing surface sampling tools were designed during the Apollo program for use in a lunar gravity environment, in contrast to the microgravity environment presented by a NEA. Among the tools required for NEA exploration is a float sample grabber, which will allow astronauts to capture and store loose rock samples from a NEA's surface without cross-contamination between collection sites. This paper describes the design, prototyping, fabrication, and testing processes for a human-operated tool for NEA float sample grabbing. Details are also given on the team's efforts to use the tool development process to inspire the next generation of engineers and scientists through a comprehensive outreach plan. Finally, qualitative and quantitative data obtained from human trials on the ground and at the NBL are presented and discussed.
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